The p53 gene is mutated in over 50 different types of human cancers, including familial and spontaneous cancers, and is believed to be the most commonly mutated gene in human cancer (Zambetti and Levine, FASEB (1993) 7:855–865; Hollstein, et al., Nucleic Acids Res. (1994) 22:3551–3555). Greater than 90% of mutations in the p53 gene are missense mutations that alter a single amino acid that inactivates p53 function. Aberrant forms of human p53 are associated with poor prognosis, more aggressive tumors, metastasis, and short survival rates (Mitsudomi et al., Clin Cancer Res 2000 October; 6(10):4055–63; Koshland, Science (1993) 262:1953).
The human p53 protein normally functions as a central integrator of signals including DNA damage, hypoxia, nucleotide deprivation, and oncogene activation (Prives, Cell (1998) 95:5–8). In response to these signals, p53 protein levels are greatly increased with the result that the accumulated p53 activates cell cycle arrest or apoptosis depending on the nature and strength of these signals. Indeed, multiple lines of experimental evidence have pointed to a key role for p53 as a tumor suppressor (Levine, Cell (1997) 88:323–331). For example, homozygous p53 “knockout” mice are developmentally normal but exhibit nearly 100% incidence of neoplasia in the first year of life (Donehower et al., Nature (1992) 356:215–221).
The biochemical mechanisms and pathways through which p53 functions in normal and cancerous cells are not fully understood, but one clearly important aspect of p53 function is its activity as a gene-specific transcriptional activator. Among the genes with known p53-response elements are several with well-characterized roles in either regulation of the cell cycle or apoptosis, including GADD45, p21/Waf1/Cip1, cyclin G, Bax, IGF-BP3, and MDM2 (Levine, Cell (1997) 88:323–331).
The family of protein arginine N-methyltransferases (PRMTs) catalyze the sequential transfer of a methyl group from S-adenosylmethionene to the side chain nitrogens of arginine residues within proteins to form methylated arginine derivatives and S-adenosyl-L-homocysteine. The methylation of arginine residues has been implicated in the regulation of signal transduction (Altschuler L et al. (1999) J. Interferon Cytokine Res. 19:189–195; Tang J et al. (2000) J. Biol. Chem. 275:19866–19876; Bedford M. T et al. (2000) J. Biol. Chem. 275:16030–16036), transcription (Chen D et al. (1999) Science 284:2174–2177), RNA transport (McBride A E et al. (2000) J. Biol. Chem. 275:3128–3136; Yun C et al. (2000) J. Cell Biol. 150:707–718), and possibly splicing (Friesen W J et al., (2001) Mol. Cell 7:1111–1117). PRMTs are conserved in evolution (Zhang X et al. (2000) EMBO J. 19:3509–3519; Weiss V H et al. (2000) Nat. Struct. Biol. 7:1165–1171).
Coactivator associated arginine Methyltransferase 1 (CARM1/PRMT4) functions in a dual role as a protein methyltransferase and a transcriptional coactivator. CARM1 interacts with the p160 coactivators to enhance nuclear receptor transcription, enhances transcription activation by the estrogen receptor, and methylates histone H3 (Chen D et al., supra). PRMT6 is the only PRMT capable of automethylation. Of the known PRMTs, CARM1 and PRMT6 localize to the nucleus (Frankel A et al. (2002) J Biol Chem. 277:3537–3543).
The ability to manipulate the genomes of model organisms such as Drosophila provides a powerful means to analyze biochemical processes that, due to significant evolutionary conservation, has direct relevance to more complex vertebrate organisms. Due to a high level of gene and pathway conservation, the strong similarity of cellular processes, and the functional conservation of genes between these model organisms and mammals, identification of the involvement of novel genes in particular pathways and their functions in such model organisms can directly contribute to the understanding of the correlative pathways and methods of modulating them in mammals (see, for example, Mechler B M et al., 1985 EMBO J 4:1551–1557; Gateff E. 1982 Adv. Cancer Res. 37: 33–74; Watson K L., et al., 1994 J Cell Sci. 18: 19–33; Miklos G L, and Rubin G M. 1996 Cell 86:521–529; Wassarman DA, et al., 1995 Curr Opin Gen Dev 5: 44–50; and Booth D R. 1999 Cancer Metastasis Rev. 18: 261–284). For example, a genetic screen can be carried out in an invertebrate model organism having underexpression (e.g. knockout) or overexpression of a gene (referred to as a “genetic entry point”) that yields a visible phenotype. Additional genes are mutated in a random or targeted manner. When a gene mutation changes the original phenotype caused by the mutation in the genetic entry point, the gene is identified as a “modifier” involved in the same or overlapping pathway as the genetic entry point. When the genetic entry point is an ortholog of a human gene implicated in a disease pathway, such as p53, modifier genes can be identified that may be attractive candidate targets for novel therapeutics.
All references cited herein, including sequence information in referenced Genbank identifier numbers and website references, are incorporated herein in their entireties.